In kidney, dopamine and DA-1 dopamine receptors regulate sodium transport; increased sodium retention can accelerate the onset of hypertension in animal models. We have recently demonstrated that defective DA-1 dopamine receptor/G protein coupling exists in renal proximal tubules (PT) of the spontaneously hypertensive rat (SHR), but not in the normotensive control rat (WKY), resulting in a loss of adenylate cyclase activity in SHR and in the ability to interact with DA- 2 dopamine receptors in membranes. The mechanisms underlying such defect couplings, which may be important in certain animal models of hypertension, will be systematically examined using biochemical and molecular approaches. The DA-1 receptors from PT of SHR and WKY will be isolated and purified and the receptors will be characterized by pharmacological and biochemical methods. The results obtained will be used to compare properties of peripheral DA-1 dopamine receptors with D-1 dopamine receptors of brain. The purified DA-1 receptors of WKY and SHR will be reconstituted with exogenous G proteins, isolated from normal and hypertensive tissue. A persistent absence of coupling will indicate whether the receptors or G proteins are defective. The ability of DA-1 receptors to couple to Gi in reconstituted systems and the affinity for a specific Gi subtype will be determined. Receptor phosphorylations will also be conducted to test if SHR receptors are desensitized. The interactions between DA-1 and DA-2 receptors will be analyzed in WKY and SHR membranes and in dispersed cells. From selective reconstitutions of crude soluble DA-1 and DA-2 receptors with exogenous G proteins, the role and identity of G proteins in mediating such interactions will be established. Using D-1 and D-5 full-length cDNA probes, the cDNA coding for the peripheral DA-1/DA-5 receptors from WKY and SHR PT cDNA libraries will be screened under low-stringency conditions. Positive clones will be rescreened using a combination of polymerase chain reaction (PCR), restriction analysis and sequencing methods. Full-length cDNA will be expressed in mammalian cells and the identity of the receptor will be verified from functional and pharmacological studies. From sequence analysis of the cloned cDNA, differences between DA-1 receptors of SHR and WKY at the genetic level will be studied. The ability of these expressed receptors to couple to G proteins will be examined. DA-1/DA-5 and DA-2 receptors may also be stably coexpressed to study their interactions.